IBN AL- HAITHAM J. FO R PURE & APPL. SC I. VO L.24 (1 ) 2011 Effect of Chilling Imbibition on Seed Germination M. K. Abbas Plant Production Department, College of Agriculture Al-Qadisiya Unive rsity Recei ved in ,2,Se pt,1995 Accepted in , 6, March ,1996 Abstract Tomato (Lycopersicon esculentum M ill) and cucumber (Cucumis sativus L.) seeds were imbided with water at 10°c or 4°c for 4, 8, and 16 hours before p lanting to st udy germination rate, mean germination time, and final germination p ercentage as well as electrolyte leakage from chilled seeds. Seeds imbided more water at 10°c comp ared to that at 4°c. Coefficient velocity of germination and final p ercent of germination decreased with the decrease of temp erature and increasing time of imbibition. Germination p ercent under laboratory conditions was higher than that under field conditions. Also, it was found that tomato seeds were more sensitive to chilling imbibition. Electrolyte leakage increased as temp erature of imbibition increases. Introduction Poor germination is a common p henomenon at sub-op timal temp eratures, which is of great concern to growers that grow seedlings in late winter and early sp ring in several regions of the world. Op timum seed germination and seedling emergence occur at relatively high temp eratures (20–30°C) for many crops such as tomato, eggp lant, bean, watermelon, cucumber, and melon [1]. Tomato and cucumber are warm season crops, therefore, they can not tolerate frost s or low temp erature damage [2]. Chilling injury occurs at temp erature range of 0-10 o c [3,4] and it causes damage to all st ages of growt h and development in p lant life cycle. Chilling imbibition is referred to the occurrence of chilling at the early st age of seed germination. It destroy s t he ability of seeds to germinate and establish the seedlings [3]. Germination is divided into three p hases: imbibition, activation and p ost - germination growt h. The largest effects of cold temp erature during germination seem to be associated to the imbibition p hase, considered the most sensitive [5]. Emergence p ercentage decreased under the st ressful temp eratures because of difficulty in water up take[6]. Cold temp erature during imbibition p hase of germination leads to the increase of escap e of solutes from the seeds, such as amino acids and carbohy drates, which has been att ributed to the disturbance of p lasma membrane integrity during imbibition at low temp erature. Cellular membrane is considered as the main target for cold damage and the p rimary cause of other metabolicdisturbances observed within cells[4]. Chillin g imbibition has been st udied in sever al crops [7, 8, 9]. In cotton, for instance, it was found t hat germination percentage was sign ificantly decreased with the increase of chillin g p eriod of two varieties [10]. Pisum germination rate and seedling growt h under low t emp erature condition were very slow compared to that at room temp erature [11, 12]. M aximum percenta ge of germinationm of So lanum nigrum was observed at 27°C while the lowest p ercentage of seed germination was observed at low t emp erature (3% after 5 day s of incubation at 5°C) [13]. Same results of low germination under low temp eratures conditions were rep orted by others (14, 15). Some invest igators have st ated that for seeds to be germin ated there should be a trigger which st imulated immediately after the hydration of the seeds under normal cond ition [16, 17]. Because temperature is one of the most effective factor controllin g seed germination [18], therefore, any unusual temperature changes will IBN AL- HAITHAM J. FO R PURE & APPL. SC I. VO L.24 (1 ) 2011 interrupt the normal series of seed germination and result in dropp ing the ability of seeds germination. Cucumb er and tomatoes are summer crop s and they are p lanted in early sp ring in order to give early p roduction, therefore, they may exp osed to low temp erature during germination. The objective of this resear ch was to determine the effect of chillin g imb ibition at its upp er limit (10 o c) and lower limit (4 o c) on seed germination of tomato and cucumber. Material and Methods This exp eriment was conducted on local varieties of tomato (Lycopersicon esculentum M ill) and cucumber (Cucumis sativus L.). Seeds of uniform size and free from any decay were chosen. Seeds were weighted and rapp ed with two lay ers of Whatman filter p aper in Petri dishs. Two group s of Petri dishs were prepared. To the first group , 10-15 ml of deionized water at 10 o c was added to each Petri dish and held in a refrigerator set to 10 o c for 4, 8, and 16 hours. For the second group , water at 4 o c was added to the Petri dish and seeds were held in a refrigerator set to 4 o c for the same p eriods of time. Control treatment was left to imbibe at room temp erature. At the end of each p eriod of time, seeds were taken and blott ed with filter p aper to remove excess of water and weighted again in order to calculate the water imbibed by seeds at each treatment. Then, seeds for each treatment were sown in Petri dishs and left under room temp erature for laboratory st udy . Three replications of 50 seeds each were used for each treatment. Number of seeds germinated was recorded daily. M ean germination time (M GT ) was calculated according to [19] as below: Σ (ni di) M GT = ------------ Σ n Σ n the total number of germinated seeds during the germination test, ni is t he number of germinated seeds on day di and di is t he number of days during the germin ation p eriod. Coefficient velocity of germination was calculated as follow [20]: A1 + A2 + ………+Ax C.V= ______________________________ X 100 A1T 1 + A2T 2 + ……….+AxTx Where A= number of seed lin gs emerged at any day T= day number Final germination p ercentage (FGP), the p ercentage of number of seeds germinated to total seeds p lanted, was obtained when exp eriment was terminated. For electrolyte leakage of chilled imbibed seeds, 1g of tomato seeds or 3g of cucumber seeds were taken immediately at the end of each p eriod of chilling at both temp eratures, soaked in 10 ml of distilled water at 25c for 24 hours and conductivity was measured [4]. For field st udy , seeds, after they were treated with chilling temp eratures at the desired p eriod of time, were sown in 3-inch diameter p ots containing a mixture of sand and loam and kept at lath house. Pot s were watered daily and M GT , C.V , and PGA were calculated in the same way for Petri dish exp eriment. A comp letely randomized design was used for both Petri dish and field exp eriments. Data were analyzed using analysis of variance and means were comp ared using Duncans new multiple range test at 5% level. Results and Discussion Results on water imbibition of seeds held at 10°c and 4°c for various p eriod of time are shown in table( 1). Generally , with the increase of time of imbibition, water up take by seeds was increased. For t omato, the amount of water taken by chilled imbided seeds was less than that for t he control. However, the p ercent of water imbibed by seeds at 4°c for 16 hour was IBN AL- HAITHAM J. FO R PURE & APPL. SC I. VO L.24 (1 ) 2011 not significantly different from that of the control. It was hy p othesized that imbibition temp erature affects seed hy dration. Higher seed water content at full hy dration was associated with lower imbibition temp eratures, app arently as a result of different hy dration rates between embry o and endosp erm[21]. Bai, [22] indicated that the greater water content of seeds imbibed at 0°c exp lains the warmer low t emp erature exothermm for seeds imbibed at lower temp eratures than those imbibed at higher temp eratures. With regard to cucumber seeds, the p ercent of water imbibed increased from 95.9% for seeds held for 4 hours at 10°c to 159.8% for seeds held for 16 hours at t he same chilling temp erature in comparison with 79.8% for the control. This increase was equal to more than double the water content of seeds of the control treatment. At 4°c, seeds have taken less water compared to that at 10°c. This may be due to a relatively warmer temp erature (10°c) and the ability of seeds coat to st retch at that temp erature. Also, it should be noted that cucumber seeds absorb much more water comp ared to tomato seeds which indicates differences in the abilities of two t issues t o take water. Germination coefficient velocity was less at 4°c in comp arison with that at 10°c for both crops and under both lab and field conditions (Table.2). It was clear that t he germination rate was decreased dramatically with the increase of time of imbibition at 4°c rather than at 10°c. Therefore, it seems that there is a time temp erature interaction in controlling the rate of germination. Under laboratory conditions, germination rates were 45.80% and 48.00% for the control and they drop p ed to 30.20% and 28.80% for seeds imbibed for 16 hours at 4°c of tomato and cucumber resp ectively. Same p att ern of germination rate was noted under field conditions. Rate of germination dropp ed from 44.10% to 28.60% for tomato and from 42.30% to 31.00% for cucumber when seeds imbibed at room temp erature and at 4°c for 16 hours resp ectively. Germination rate, and consequently germination index, is imp ortant for crop establishment. They are related to a high seed vigor and this may be the cause of the bett er p erformance of crops [23]. This result comes in agreement with the results of [11, 12] who mentioned that Pisum germination rate and seedling growt h under low temp erature condition were very slow compared to that at room temp erature. Also, the results of (8) who found that imbibition temperature at 5°c in tomato affected the rate of germination which decline across the different moist ure contents. M ean germination time revealed an op p osite p att ern to germination coefficient velocity (Table. 3). M GT increased as temp erature decreased and time of imbibition increased. This result may indicate an inhibition in the metabolic activities at low temp erature (4). Chen, [24] observed that the p eriod of greatest sensitivity to cold corresp onds t o the first 30 min of imbibition which affect all the consequence events in seed germination and seedling establishment. Final germination percentage (FGP) decreased also with the decrease of temp erature and the increase of time of imbibition (Table 4). FGP under laboratory conditions was higher than that under field conditions. This may be due to the favorable conditions at laboratory rather than at the field. The lowest p ercentage was obtained at 4°c for 16 hour of imbibition for both tomato and cucumber under laboratory and field conditions. Therefore, it seems that two factors controlling the p ercentage of germination; temp erature and time of imbibition. Under laboratory conditions, tomato germination p ercentage dropp ed from 99.30% to 73.20%, and cucumber germination percentage drop p ed from 98.20% to 80.60% for seeds imbibed at room temp erature and 4°c (16h), resp ectively. Same trend was found under field conditions. This direction of decreasing p ercentage of germination with the decrease of temp erature and increase of time of imbibition is corresp ondence to that for the rate of germination. Levitt [4] has att ributed the decrease in rate and p ercentage of germination to the overall decrease in metabolic activities at chilling temp eratures. Also, it was found that imbibition at low temp eratures has a harmfull effect on cell membranes which leads to leakage of cell contents and then decrease seed germinability [3]. Pea seeds have showed reduced germination p ercentage when the IBN AL- HAITHAM J. FO R PURE & APPL. SC I. VO L.24 (1 ) 2011 imbibition and germination were carried out under chilling st ress on comp arison with t he seeds germinated under room temp erature condition [12]. Poor survival of seeds and seedlings and high leakage of organic and inorganic substances were commonly observed as a result of imbibition at chilling st ress [25]. Berrie, [26] indicated that during imbibition under normal temp erature, range of dry seeds (less than 20% water content) membranes p hosp holip ids will rearrange themselves from hexagonal to lamellar shap e but at chilling temp erature, this rearrangement will p ermit the formation of channels or p ores at t he membranes and therefore increases the leakage of cell contents and as a result effect all biological functions of t he membrane. It is clear from the results in table 5, that the electrolyte leakage from seeds exp osed to 4°c was more than that at 10°c. Chen, [24] has st ated that the decrease in exp osing temp eratures cause leaching of several imp ortant cellular contents from the cells due to membrane injury that intensifies the damage to germinating seed. It was also known that imbibition phase was relatively more sensitive to chilling as indicated by the relatively higher damage to membranes [27]. The results show obviously that p rolong of exp osing time of seeds to chilling temp erature caused a continuous increase in leachates. This result comes in agreement with the result of [7] who mentioned that chilling of wheat seeds for 96 h showed maximum electrical conductivity in comparison with that for 1, 2, 6 and 12 h after imbibition. The leakage of solutes from seeds during imbibition is due to reorganization or repair of membrane comp onents and conformational changes occurring in cell membranes [28,29]up on dry ing of seeds, or may be due to membrane deterioration [30]. It was suggested that a sp ecific structure of the seed coat called semip ermeable lay er would p lay a significant role in regulating the electrolyte leakage[8]. However it is wort h to mention that tomato seeds were more sensitive to chilling than cucumber as indicated by the differences in the amount of leachates after exp osing to chilling temp eratures. From the results p resented here, we can conclude that rate and p ercentage of seed germination were less at 4°c comp ared to that at 10°c under laboratory and field conditions. Also, these two p arameters were more effected under field rather than laboratory conditions. Increase of time of imbibition decreased both rate and p ercent of seed germination. Tomato seeds were more sensitive to chilling imbibition than cucumber seeds. Re ferences 1. Tzortzakis, N.G.( 2009). Effect of p re-sowing treatment on seed germination and seedling vigor in endive and chicory . Hort Sci . (Prag u e). 36(3): 117–125. 2. Breidenbach, R.W., Waring, A. C. (1977). Resp onse to chilling of tomato 192.-:19060seedlings and cell susp ension cultured cells. Plant Physiol. 3. Lyons, J.H.( 1973). Chilling Injury in Plants. Ann. Rev. Plant Phy siol. 466.-:44524 4. Levitt , J.( 1980). Resp onses of Plants to Environmental Stresses, vol.1. Chillin g, Fre ezing and High Temp erature Stresses. Academic Press. New York. 5. Blum, A. (1988). Plant Breeding for Stress Environments. P 99-132: Cold Resist ance. Boca Raton: CRC. 6. Arin, L., and Kiy ak, Y.( 2003). The effect of p re-sowing on emer gence and seed lin g growt h of tomato seed (Lycopersicon esculentum M ill.) under several stress conditions. Pakistan J. Biol. Sci. 6(11) : 990-994. 7. Khani, M . B., Ghurchani , M . Hussain, M . and M ahmood ,A. ( 2010). Wheat seed invigoration by p re-sowing chillin g treatments. Pakistan J. Bot., 42(3): 1561-1566. 8. Chachalis, D.; Darawsheh , M.K. and Khah,E.M.( 2008). Effect s of init ial seed moisture content, imbibit ion t emperature and seed vigor on germin at ion, elect rolyt e leak age and seedling gro wt h in plum tomato es. Foo d, Agric. Envir. 6 (3, 4) : 299-304. 9. Bithell, M cKenzie,S.L., Bourdot, B. A. Hill, G. D. and Wratt en, S. D. (2002). Germination requir ements of laboratory st ored seeds of Solanum nigrum and Solanum physalifolium. New Z ealand Plant Prot. 55:222-227. IBN AL- HAITHAM J. FO R PURE & APPL. SC I. VO L.24 (1 ) 2011 10. Anjum, Z. I., and Khatoo, A.( 2003). Chilling effect on germination and 299.-:2972(3)m. Asian J. Plant Sci. Gossypiuseedling vigour of som e sp ecies of 11. Shereena, J., and Salim, N. (2006) a. Influence of seed moist ure content and leakage on germination and viability in Pisum sativum L. seeds. Int . J. Bot any 2 (4):427-430. 12. Shereena, J., and N. Salim. 2006b. Chilling tolerance in Pisum sativum L.: 1050.-):10475(6An ecolo gical ad aptation. Asian J. Plant Phy siol. 13. Suthar, C., Naik, V.R. and M ulani, R.M .( 2009). Seed and seed germination in So lanum nigrum Linn. American-Eurasian J. Agric. Envir. Sci., 5 (2): 179-183. 14. Schulze, D., Hopp er,N. J. Ganaraway and Jividen ,G. (1997). Evaluation of chillin g tolerance in cotton genoty p es. Proc. Beltwide Cott on Conf. 2:745-750. 15. Baloch, M .J., Lakho, R. Bhutt o, H.U. and Baloch ,A.H.( 1999). Genotype X environment iteraction analy sis of cotton varieties (Gossypium hirsutum L). Sindh, Biological, J. Plant Sci. 1:1-6. 16. Salisbury , P.B., and Ross C.W.( 1986). Plant Phy siology . California. 540pp . 17. Berry, J.A., and Raison, J.R. 1981. Resp onse of macrophytes to temp erature. 726.-: 72312AEncy clop edia of Plant Physiol. 18. Shoemaker, C.A., and Car lson, W.H. 1992. Temp erature and light affect (2):181. 27cultorum. HortSci. -seed germin ation of begonia sa mperflorens 19. Youshen g, C. and Sziklai ,O.(1985). Prelimin ary st udy on the germination of Toona sinensis (A. Juss.) Roem. seed from eleven Ch inese p rovenances. For. Eco l. M anage. 10: 269- 281. 20. M ohammed, A. A. K. 1985. Plant Phy siology . 3. M ousel. Iraq. 21. Bai, Y., D. T . Booth, and J.T. Romo. 1999. imbibition temp erature affects winterfat (Eurotia lanata) (Pursh) M oq.) seed hydration and cold hardiness resp onse. J. Range M anage. 52:271-274. 22. Bai, Y., Booth, D.T . and Romo, J.T . (1998). Winterfat (Eurotia lanata (Pursh) M oq.) seedbed eco logy : Low t emp erature exotherms and cold-h ardiness in hy drated seeds as influenced by imbibition temperature. Ann. Bot. 81:598-602. 23. Pereira, R., and S. C. M ilach. 2004. Cold tolerance at t he germination st age of rice: M ethods of evaluation and characterization of genoty p es. Sci. Agric. (Pir acicaba, Braz.), 61(1):1-8. 24. Chen T., Yamamoto S. D., Gust a L.V. and Slinkard, A. E.(1983). Imbibitional chilling injury during chickp ea germination. J. Amer. So c. Hort. Sci. 108:944-948. 25. Thornton, J.M ., Powell, A.A. and M attews ,S.( 1990). Invest igation of the relationship between seed leachate conductivity and germination of Brassica seeds. Ann. 135.-:129117Biol., App lied 26. Berrie, A.M . 1984. Germination and Dormancy. In: Wilk ins, M .B. (ed.).1984. Advance Plant Phy siology . London. Pitt man. 514 pp . 27. Nayyar, H., K. Gurinder and C. Subas b. (2004 ). Response of chickpea seed germ ination to spermidine treat ment to overcome co ld injury. Inter. Chickpea and P igeonpea Newsl. 11:25-28. 28. Senar athna, T., Gusses ,J.F. and M cKersie ,B.D. (1988). M embrane chan ges in ageing soy bean axes. Plant Phy siol., 73:85-91. 29. Bewley , J.D., 1997. Seed germination and dormancy. Plant Cell. 9:1055-1066. 30. Farooq, M ., Basra, S. M . Hafeez, K. and Warriach, E.A. (2004). Influ ence of high and low temp erature treatments on seed germination and seed lin gs vi gor of coarse and fine rice. Int. Rice Res. Newsl. 29(2):75-7 IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.24 (1) 2011 Table( 1) Percent of water imbibed by tomato and cucum ber seeds at 10°c and 4°c for various periods of ti me. Wat er imbibed T reat ment Cucum ber Tomato Imbibit ion t ime (h) T emp erat ure 79.80 d 50.20 z a cont rol 95.90 c 34.50 d 4 10 °c 127.40 b 42.10 bc 8 159.80 a 47.30 ab 16 79.80 d 37.30 cd 4 4°c 124.50 b 37.30 cd 8 109.10 bc 49.00 a 16 Z mean separation within column, b y Duncans new multiple range test, at 5% level - numbers f ollowed by the same letter(s ) are n ot sign ificantly different. Table( 2) Germination coefficient velocity of tomato and cucumber seeds imbibed at 10°c and 4°c for various period of time. Germination coeff icient velocity Treatment Cucumber L F Tomato L F Imbibition time (h) Temp erature 42.30c 48.00a 44.10a 45.80 z a control 47.10b 46.00a 42.00ab 33.30d 4 10°c 52.10a 40.00b 39.30c 42.00b 8 47.50b 36.00cd 40.90bc 40.10b 16 42.40c 38.30bc 39.20c 34.90cd 4 4°c 35.00d 35.10d 31.20e 36.00c 8 31.00e 28.80e 28.60f 30.20e 16 Z mean separation within column, by Duncans new multiple range test, at 5% level L Laboratory condition F Field condition - numbers followed by the same letter(s) are not sign ificantly different. Table( 3) Mean germinati on ti me of tomato and cucumber seeds imbibed at 10°c and 4°c for various period of time. M ean germination time Treatment Cucumber L F Tomato L F Imbibition time (h) Temp erature 6.75e 6.05d 7.85d 6.72 z d control 6.98de 6.00d 7.90d 7.12c 4 10°c 7.43cd 6.78c 8.40c 7.10c 8 7.89bc 7.85ab 8.95b 6.90c 16 7.80bc 7.55b 8.53bc 7.12c 4 4°c 8.30b 7.50b 9.11b 7.85b 8 8.95a 8.21a 10.09a 8.55a 16 Z mean separation within column, b y Duncans new multiple range test, at 5% level L Laboratory condition F Field condition - nu mbers followed by the same letter(s) are n ot sign ificantly different. IBN AL- HAITHAM J. FO R PURE & APPL. SC I. VO L.24 (1 ) 2011 Table( 4) Final germination percentage (FGP) of tomato and cucumber imbibed at 10°c and 4°c for various periods of ti me. Final germination p ercentage (FGP) Treatment Cucumber L F Tomato L F Imbibition time (h) Temp erature 90.10 a 98.20 a 86.30 a 99.30 z a control 89.10 a 92.40 a 86.20 a 93.30 a 4 10°c 87.20 a 95.10 a 82.20 ab 90.00 ab 8 83.40 ab 88.70 b 77.20 b 88.90 b 16 86.70 a 92.50 a 81.70 ab 95.20 a 4 4°c 77.50 b 79.60 c 75.40 b 98.00 a 8 70.60 c 80.60 c 68.50 c 73.20 c 16 Z mean separation within column, by Duncans new multiple range test, at 5% level L Laboratory condition F Field condition - numbers followed by the same letter(s) are not sign ificantly differe Table(5) Electrolyte leakage from seeds of tomato and cucumber chi lle d at 10°c and 4°c for various period of time. Electrolyte leakage Treatment Cucumber Tomato Imbibition time (h) Temp erature 18.56 e 22.20 z f control 21.75 e 27.50 e 4 10°c 26.66 c 33.45 d 8 34.95 b 44.80 b 16 27.80 c 39.33 c 4 4°c 37.65 b 46.70 b 8 45.45 a 57.55 a 16 Z mean separation within column, b y Duncans n ew multiple range test, at 5% level - numbers followed b y the same letter(s ) are n ot significantly different. 2011) 1( 24المجلد مجلة ابن الهیثم للعلوم الصرفة والتطبیقیة نسبة انبات البذور فيتأثیر التشرب بدرجات الحرارة المنخفضة مجید كاظم عباس جامعة القادسیة ،كلیة الزراعة،قسم االنتاج النباتي 1995،ایلول ،2استلم البحث في 1996، اذار،6قبل البحث في الخالصة Lycopersicon esculentum M)شـبعت بـذور الطماطـة ill) والخیـار(Cucumis sativus L.) بالمـاء بدرجـة . سـاعة قبـل زراعتهـا تحـت ظـروف المختبـر فـي اطبـاق بتـري او فـي الظلـة الخشـبیة 16او 8و 4 مـددل 10cاو 4cحـرارة نسبة االنبات وكذلك كمیة المواد االلكترولیتیة الناضحة من البذور بعـد و،معدل عدد ایام االنبات و،قیست سرعة االنبات مقارنــة 10cاوضــحت النتـائج ان البــذور قـد تشــبعت بكمیـة مــاء اكبـر بدرجــة .تعریضـها للتشــبع بـدرجات الحــرارة المنخفضـة سرعة االنبات قلت مع خفـض كما ان نسبة و . وان بذور الطماطم كانت اقل قابلیة الخذ الماء من بذور الخیار. 4cبدرجة درجـة حـرارة التشــرب وزیـادة مــدة التشـرب فـي حــین زادت كمیـة المــواد االلكترولیـة الناضـحة مــن البـذور مــع خفـض الحــرارة كـذلك فـان بــذور . نسـبة االنبـات تحــت ظـروف المختبـر كانـت اعلـى مقارنـة بتلــك تحـت ظـروف الحقـل. وزیـادة مـدة التشـرب .للتشرب بدرجات الحرارة المنخفضة الطماطم كانت اكثر حساسیة